The evolutionary legacy of immune checkpoint inhibitors.

Immune check point inhibitors (ICIs) have marked their existence in the field of cancer immunotherapy. Their existence dates to 2011 when the first anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) got its FDA approval for the management of metastatic melanoma. The class of ICIs now also include antibodies against programmed cell death-1 (PD-1) and its ligand (PD-L1) which immediately gained FDA approval for use against multiple cancer types because of their effect on patient survival. These discoveries were followed by a significant rise in the identification of novel ICIs with potential anti-tumor response. Researchers have identified various novel checkpoint inhibitors which are currently under clinical trials. Despite the success of ICIs, only a small subset of patients with specific tumor types achieves a promising response. Not only efficient therapeutic response but also development of resistance, recurrence and other immune-related adverse effects limit the applicability of immune checkpoint inhibitors. These challenges can only be addressed when a directed approach is implemented at both basic and translational level. In this review, we have briefly discussed the history of ICIs, the next generation of inhibitors which are currently under clinical trial and mechanisms of resistance that can lead to treatment failure. Ultimately, by combining these insights researchers might be able to achieve a more durable and effective response in cancer patients.

Immune checkpoint proteins: Signaling mechanisms and molecular interactions in cancer immunotherapy.

Immune checkpoint proteins (ICP) are currently one of the most novel and promising areas of immune-oncology research. This novel way of targeting tumor cells has shown favorable success over the past few years with some FDA approvals such as Ipilimumab, Nivolumab, Pembrolizumab etc. Currently, more than 3000 clinical trials of immunotherapeutic agents are ongoing with majority being ICPs. However, as the number of trials increase so do the challenges. Some challenges such as adverse side effects, non-specific binding on healthy tissues and absence of response in some subset populations are critical obstacles. For a safe and effective further therapeutic development of molecules targeting ICPs, understanding their mechanism at molecular level is crucial. Since ICPs are mostly membrane bound receptors, a number of downstream signaling pathways divaricate following ligand-receptor binding. Most ICPs are expressed on more than one type of immune cell populations. Further, the expression varies within a cell type. This naturally varied expression pattern adds to the difficulty of targeting specific effector immune cell types against cancer. Hence, understanding the expression pattern and cellular mechanism helps lay out the possible effect of any immunotherapy. In this review, we discuss the signaling mechanism, expression pattern among various immune cells and molecular interactions derived using interaction database analysis (BioGRID).

GABAA receptor agonist suppresses pediatric medulloblastoma progression by inhibiting PKA-Gli1 signaling axis.

The Sonic hedgehog-activated subgroup of medulloblastoma (SHH-MB) is one of the most common malignant pediatric brain tumors. Recent clinical studies and genomic databases indicate that GABAA receptor holds significant clinical relevance as a therapeutic target for pediatric MB. Herein, we report that "moxidectin," a GABAA receptor agonist, inhibits the proliferation of Daoy, UW426, UW228, ONS76, and PFSK1 SHH-MB cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy demonstrated that moxidectin significantly induced GABAA receptor expression and inhibited cyclic AMP (cAMP)-mediated protein kinase A (PKA)-cAMP response element-binding protein (CREB)-Gli1 signaling in SHH-MB. Gli1 and the downstream effector cancer stem cell (CSC) molecules such as Pax6, Oct4, Sox2, and Nanog were also inhibited by moxidectin treatment. Interestingly, moxidectin also inhibited the expression of MDR1. Mechanistic studies using pharmacological or genetic inhibitors/activators of PKA and Gli1 confirmed that the anti-proliferative and apoptotic effects of moxidectin were mediated through inhibition of PKA-Gli1 signaling. Oral administration of 2.5 mg/kg moxidectin suppressed the growth of SHH-MB tumors by 55%-80% in subcutaneous and intracranial tumor models in mice. Ex vivo analysis of excised tumors confirmed the observations made in the in vitro studies. Moxidectin is an FDA-approved drug with an established safety record, therefore any positive findings from our studies will prompt its further clinical investigation for the treatment of MB patients.

Pimavanserin: A Novel Autophagy Modulator for Pancreatic Cancer Treatment.

Pancreatic tumors exhibit high basal autophagy compared to that of other cancers. Several studies including those from our laboratory reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we evaluated the autophagy and apoptosis inducing effects of Pimavanserin tartrate (PVT). Autophagic effects of PVT were determined by Acridine Orange assay and Transmission Electron Microscopy analysis. Clinical significance of ULK1 in normal and pancreatic cancer patients was evaluated by R2 and GEPIA cancer genomic databases. Modulation of proteins in autophagy signaling was assessed by Western blotting and Immunofluorescence. Apoptotic effects of PVT was evaluated by Annexin-V/APC assay. Subcutaneous xenograft pancreatic tumor model was used to evaluate the autophagy-mediated apoptotic effects of PVT in vivo. Autophagy was induced upon PVT treatment in pancreatic ducal adenocarcinoma (PDAC) cells. Pancreatic cancer patients exhibit reduced levels of autophagy initiator gene, ULK1, which correlated with reduced patient survival. Interestingly, PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, LC3A/B, and cleavage of caspase-3, an indicator of apoptosis in several PDAC cells. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in PDAC cells. Notably, chloroquine abrogated the growth suppressive effects of PVT by 25% in BxPC3 tumor xenografts in nude mice. Collectively, our results indicate that PVT mediated pancreatic tumor growth suppression was associated with induction of autophagy mediated apoptosis.

Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells.

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-ß and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-ß, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-ß and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

Drug rechanneling: A novel paradigm for cancer treatment.

Cancer continues to be one of the leading contributors towards global disease burden. According to NIH, cancer incidence rate per year will increase to 23.6 million by 2030. Even though cancer continues to be a major proportion of the disease burden worldwide, it has the lowest clinical trial success rate amongst other diseases. Hence, there is an unmet need for novel, affordable and effective anti-neoplastic medications. As a result, a growing interest has sparkled amongst researchers towards drug repurposing. Drug repurposing follows the principle of polypharmacology, which states, "any drug with multiple targets or off targets can present several modes of action". Drug repurposing also known as drug rechanneling, or drug repositioning is an economic and reliable approach that identifies new disease treatment of already approved drugs. Repurposing guarantees expedited access of drugs to the patients as these drugs are already FDA approved and their safety and toxicity profile is completely established. Epidemiological studies have identified the decreased occurrence of oncological or non-oncological conditions in patients undergoing treatment with FDA approved drugs. Data from multiple experimental studies and clinical observations have depicted that several non-neoplastic drugs have potential anticancer activity. In this review, we have summarized the potential anti-cancer effects of anti-psychotic, anti-malarial, anti-viral and anti-emetic drugs with a brief overview on their mechanism and pathways in different cancer types. This review highlights promising evidences for the repurposing of drugs in oncology.

Pimozide Suppresses the Growth of Brain Tumors by Targeting STAT3-Mediated Autophagy.

Brain tumors are considered as one of the most aggressive and incurable forms of cancer. The majority of the patients with brain tumors have a median survival rate of 12%. Brain tumors are lethal despite the availability of advanced treatment options such as surgical removal, chemotherapy, and radiotherapy. In this study, we have evaluated the anti-cancer effects of pimozide, which is a neuroleptic drug used for the treatment of schizophrenia and chronic psychosis. Pimozide significantly reduced the proliferation of U-87MG, Daoy, GBM 28, and U-251MG brain cancer cell lines by inducing apoptosis with IC50 (Inhibitory concentration 50) ranging from 12 to 16 µM after 48 h of treatment. Our Western blotting analysis indicated that pimozide suppressed the phosphorylation of STAT3 at Tyr705 and Src at Tyr416, and it inhibited the expression of anti-apoptotic markers c-Myc, Mcl-1, and Bcl-2. Significant autophagy induction was observed with pimozide treatment. LC3B, Beclin-1, and ATG5 up-regulation along with autolysosome formation confirmed the induction of autophagy with pimozide treatment. Inhibiting autophagy using 3-methyladenine or LC3B siRNA significantly blocked the apoptosis-inducing effects of pimozide, suggesting that pimozide mediated its apoptotic effects by inducing autophagy. Oral administration of 25 mg/kg pimozide suppressed the intracranially implanted U-87MG tumor growth by 45% in athymic nude mice. The chronic administration of pimozide showed no general signs of toxicity, and the behavioral activity of the mice remained unchanged. Taken together, these results indicate that pimozide inhibits the growth of brain cancer by autophagy-mediated apoptosis.

Cancer cells stemness: A doorstep to targeted therapy.

Recent advances in research on cancer have led to understand the pathogenesis of cancer and development of new anticancer drugs. Despite of these advancements, many tumors have been found to recur, undergo metastasis and develop resistance to therapy. Accumulated evidences suggest that small population of cancer cells known as cancer stem cells (CSC) are responsible for reconstitution and propagation of the disease. CSCs possess the ability to self-renew, differentiate and proliferate like normal stem cells. CSCs also appear to have resistance to anti-cancer therapies and subsequent relapse. The underlying stemness properties of the CSCs are reliant on multiple molecular targets such as signaling pathways, cell surface molecules, tumor microenvironment, apoptotic pathways, microRNA, stem cell differentiation, and drug resistance markers. Thus an effective therapeutic strategy relies on targeting CSCs to overcome the possible tumor relapse and chemoresistance. The targeted inhibition of these stem cell biomarkers is one of the promising approaches to eliminate cancer stemness. This review article summarizes possible targets of cancer cell stemness for the complete treatment of cancer.

Role of Phytochemicals in Cancer Prevention.

The use of synthetic, natural, or biological agents to minimize the occurrence of cancer in healthy individuals is defined as cancer chemoprevention. Chemopreventive agents inhibit the development of cancer either by impeding DNA damage, which leads to malignancy or by reversing or blocking the division of premalignant cells with DNA damage. The benefit of this approach has been demonstrated in clinical trials of breast, prostate, and colon cancer. The continuous increase in cancer cases, failure of conventional chemotherapies to control cancer, and excessive toxicity of chemotherapies clearly demand an alternative approach. The first trial to show benefit of chemoprevention was undertaken in breast cancer patients with the use of tamoxifen, which demonstrated a significant decrease in invasive breast cancer. The success of using chemopreventive agents for protecting the high risk populations from cancer indicates that the strategy is rational and promising. Dietary components such as capsaicin, cucurbitacin B, isoflavones, catechins, lycopenes, benzyl isothiocyanate, phenethyl isothiocyanate, and piperlongumine have demonstrated inhibitory effects on cancer cells indicating that they may serve as chemopreventive agents. In this review, we have addressed the mechanism of chemopreventive and anticancer effects of several natural agents.

CRISPR-Cas9: A multifaceted therapeutic strategy for cancer treatment.

CRISPR-Cas9 is an RNA guided endonuclease that has revolutionized the ability to edit genome and introduce desired manipulations in the target genomic sequence. It is a flexible methodology and is capable of targeting multiple loci simultaneously. Owing to the fact that cancer is an amalgamation of several genetic mutations, application of CRISPR-Cas9 technology is considered as a novel strategy to combat cancer. Genetic and epigenetic modulations in cancer leads to development of resistance to conventional therapy options. Given the abundance of transcriptomic and genomic alterations in cancer, developing a strategy to decipher these alterations is critical. CRISPR-Cas9 system has proven to be a promising tool in generating cellular and animal models to mimic the mutations and understand their role in tumorigenesis. CRISPR-Cas9 is an upheaval in the field of cancer immunotherapy. Furthermore, CRISPR-Cas9 plays an important role in the development of whole genome libraries for cancer patients. This approach will help understand the diversity in genome variation among the patients and also, will provide multiple variables to scientists to investigate and improvise cancer therapy. This review will focus on the discovery of CRISPR-Cas9 system, mechanisms behind CRISPR technique and its current status as a potential tool for investigating the genomic mutations associated with all cancer types.

Role of Forkhead Box Class O proteins in cancer progression and metastasis.

It is now widely accepted that several gene alterations including transcription factors are critically involved in cancer progression and metastasis. Forkhead Box Class O proteins (FoxOs) including FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX and FoxO6 transcription factors are known to play key roles in proliferation, apoptosis, metastasis, cell metabolism, aging and cancer biology through their phosphorylation, ubiquitination, acetylation and methylation. Though FoxOs are proved to be mainly regulated by upstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt signaling pathway, the role of FoxOs in cancer progression and metastasis still remains unclear so far. Thus, with previous experimental evidences, the present review discussed the role of FoxOs in association with metastasis related molecules including cannabinoid receptor 1 (CNR1), Cdc25A/Cdk2, Src, serum and glucocorticoid inducible kinases (SGKs), CXCR4, E-cadherin, annexin A8 (ANXA8), Zinc finger E-box-binding homeobox 2 (ZEB2), human epidermal growth factor receptor 2 (HER2) and mRNAs such as miR-182, miR-135b, miR-499-5p, miR-1274a, miR-150, miR-34b/c and miR-622, subsequently analyzed the molecular mechanism of some natural compounds targeting FoxOs and finally suggested future research directions in cancer progression and metastasis.